Methods and devices for detecting non-complexed prostate specific antigen

ABSTRACT

The present invention relates to novel methods and devices for detecting non-complexed prostate specific antigen (PSA), which can be used either alone or in conjunction with total PSA tests to identify patients having either benign prostatic diseases (BPD), such as benign prostatic hyperplasia, prostatitis, or glandular atrophy or prostatic adenocarcinoma (CAP). In a biological sample, one can find not only non-complexed PSA, but also PSA which has formed a complex with α 1 -antichymotrypsin (ACT). The present invention removes or precipitates complexed PSA (PSA-ACT) and ACT from a fluid sample, thereby removing any possible interference due to the binding of complexed PSA to assay reagents. The method requires contacting a biological fluid sample possibly containing a mixture of complexed PSA and non-complexed PSA either with an immuno-precipitating reagent or a device having attached an ACT specific binding partner which in both cases specifically binds only to ACT and the ACT portion of complexed PSA, thereby precipitating ACT and the bound complexed PSA and, thus, leaving any non-complexed PSA unbound and in solution. Then, the sample is measured for non-complexed PSA by means of a conventional specific binding reaction. The device can be a filter having a modified filter media that binds ACT and complexed PSA, while permitting the sample to flow or be pulled through the filter media and into assay reagents. Alternatively, the device can be a removable media that is placed into a sample, allowed to remain long enough to bind any ACT and complexed PSA present, and then withdrawn from the sample prior to running a PSA specific binding assay.

RELATED APPLICATION

[0001] The present specification is a continuation-in-part of Ser. No.08/918,839, filed Aug. 26, 1997.

TECHNICAL FIELD

[0002] The present invention relates to novel methods and devices fordetecting non-complexed prostate specific antigen (PSA), which can beused either alone or in conjunction with total PSA tests to identifypatients having either benign prostatic diseases (BPD), such as benignprostatic hyperplasia, prostatitis, or glandular atrophy or prostaticadenocarcinoma (CAP). In a biological sample, one can find not onlynon-complexed PSA, but also PSA which has formed a complex withα1-antichymotrypsin (ACT). The present invention removes or precipitatescomplexed PSA (PSA-ACT) and ACT from a fluid sample, thereby removingany possible interference due to the binding of complexed PSA to assayreagents. The method requires contacting a biological fluid samplepossibly containing a mixture of complexed PSA and non-complexed PSAeither with an immuno-precipitating reagent or a device having attachedan ACT specific binding partner which in both cases specifically bindsonly to ACT and the ACT portion of complexed PSA, thereby precipitatingACT and the bound complexed PSA and, thus, leaving any non-complexed PSAunbound and in solution. Then, the sample is measured for non-complexedPSA by means of a conventional specific binding reaction. The device canbe a filter having a modified filter media that binds ACT and complexedPSA, while permitting the sample to flow or be pulled through the filtermedia and into assay reagents. Alternatively, the device can be aremovable media that is placed into a sample, allowed to remain longenough to bind any ACT and complexed PSA present, and then withdrawnfrom the sample prior to running a PSA specific binding assay.

BACKGROUND ART

[0003] PSA is recognized as a molecular marker for CAP. Blood or serumbased immunoassays measuring the total PSA level have been commerciallyavailable for a number of years. However, the detection of total PSAdoes not necessarily mean that a patient has CAP. In order todistinguish CAP, a total PSA test has to satisfy two elements: a highsensitivity—the ability to detect disease when present, and a highspecificity—the ability to detect true negatives and avoid falsepositives. From clinical experience, total PSA tests have becomegenerally accepted as being predictive of CAP if the total PSA level isgreater than 10.0 ng/ml. Total PSA values between 0.0 ng/ml and about3.9 ng/ml have been considered generally predictive of no disease beingpresent, with a value of about 3.5 ng/ml being used for men under 60years old and about 2.5 ng/ml being used for men under 50 years old.(See Oesterling, J. E., Cooner, W. H., Jacobsen, S. J., Guess H. A., andLieber, M. M.: “Influence of Patient Age on the Serum PSA Concentrationand Important Clinical Observations”: Urol. Clin. North Am.; Vol. 20:671-680, 1993.)

[0004] PSA is primarily organ-specific, not cancer specific. Thus, PSAin blood or serum can result not only from CAP, but also from normal orhyperplastic prostate tissues. Historically, a total PSA test cannotreliably distinguish BPD from CAP at less than 10.0 ng/ml. Studies havefound that 43% (136/319) of patients with organ-confined CAP have atotal PSA value within the normal range of less than 4.0 ng/ml.Moreover, about 25% (148/597) of men with BPD have a total PSA valueabove 4.0 ng/ml. (See Oesterling, J. E.: “Prostate Specific Antigen: ACritical Assessment of the Most Useful Tumor Marker for Adenocarcinomaof the Prostate”, J. Urol., Vol:145: 907-923, 1991.) Standard medicalpractice is to biopsy patients over 60 years old having total PSA levelsof between 4.0 ng/ml and 10.0 ng/ml because about 30% of those patientshave CAP. Likewise, patients between 50 years and 60 years old whosetotal PSA falls between 3.5 ng/ml and 10.0 ng/ml and patients under 50years old whose total PSA falls between 2.5 ng/ml and 10.0 ng/ml arealso biopsied under current medical practice.

[0005] One method for detecting CAP is disclosed in U.S. Pat. No.5,501,983 to Hans Lilja et alia. In general, the Lilja patent disclosesusing immunoassays to measure “free PSA” and a complexed form of PSA.“Free PSA” is a 33 kDa single chain glycoenzyme that is produced by theepithelial cells lining the acini and prostatic ducts of the prostategland. Complexed PSA refers primarily to a 90 kDa complex of PSA boundto α1-antichymotrypsin (ACT) protein. Free PSA and complexed PSA, andtheir proportions are applied in the diagnosis of patients with CAP.Throughout, the specification discloses using a combination of a freePSA to total PSA (F/T) proportion and a complexed PSA to total PSA (C/T)proportion for use in diagnosing CAP. No prostate needle biopsy wereperformed on the patients, and the patients covered a full range oftotal PSA values. The text provides no guidance as to specifically howone uses these proportions.

[0006] A significant advance in diagnosing BPD in a male human patientwithout requiring a biopsy is disclosed by Luderer, A. A., et alia in“Measurement of the Proportion of Free to Total Prostate-SpecificAntigen Improves Diagnostic Performance of Prostate-Specific Antigen inThe Diagnostic Gray Zone of Total Prostate-Specific Antigen”, Urol.,Vol. 46: 187-194, 1995. This reflex method eliminates the need for aboutone-third of those patients to undergo such a biopsy. For those patientsin the gray diagnostic zone, the method comprises four steps. First, onemeasures the total PSA level in the blood or serum of the patient.Second, one measures the free PSA level in the blood or serum of apatient, but only if he has a total PSA level of between about 2.5 ng/mland about 10.0 ng/ml. If the patient has a total PSA level below 2.5ng/ml, then he is diagnosed to have BPD. If the patient has a total PSAlevel above 10.0 ng/ml, then he is presumed to have CAP and must bebiopsied. Third, one calculates the proportion of free PSA to total PSA.Fourth and finally, one diagnoses the patient as having BPD if thecalculated proportion of free PSA to total PSA is equal to or greaterthan about 25%.

[0007] Another method for detecting complexed PSA has been proposed byYeung, K. K., et alia, in “Novel Immunoassay for the Measurement ofComplexed Prostate-Specific Antigen in Serum”, Clin. Chem., Vol.44:1216-1223, 1998. In this assay an antibody is used to bind to aparticular epitope, rendering a potential assay interferent unable to bedetected by immunoassay antibodies. In particular, antibodies specificfor the region on PSA where ACT binds to PSA are used to change theconformation of non-complexed PSA, thereby rendering the non-complexedPSA unable to be detected by immunoassay antibodies that can bind onlyto complexed PSA. Only antibodies which cause the desired conformationalchange will work.

[0008] The removal of an unwanted component from a biological sample isdisclosed in U.S. Pat. No. 5,403,745 to James F. Ollington et alia. Whenassaying for a cholesterol analyte in a targeted lipoprotein class,there can be present in a sample at least one cholesterol-containinginterfering substance in another lipoprotein class. This interferingsubstance is removed by binding the interfering substance withimmobilized antibodies.

DISCLOSURE OF THE INVENTION

[0009] The present invention relates to a method for detectingnon-complexed PSA in a biological sample using an assay thatincorporates a specific binding reaction. For the purposes of thisinvention, a distinction is made between “free PSA” and “non-complexedPSA”. “Free PSA” is defined as the form of PSA that is measured byantibodies disclosed by Lilja, namely, those which will normallyrecognize an epitope on PSA when PSA is not complexed with ACT, but willnot recognize that same epitope if the PSA is complexed with ACT.“Non-complexed PSA” is the PSA moiety which remains after all ACT moietyin a mixture of free PSA and complexed PSA has been specifically boundby an antibody and removed or precipitated. The distinction between thetwo is important. In some cases, the Lilja method can detect as free PSAwhat is really complexed PSA. This erroneous detection occurs because insome samples the ACT binding epitopes on PSA are revealed due toconformational changes induced perhaps by sample specific interferents.The free PSA antibody can now detect the PSA portion of the complexedPSA. This over recovery of signal for free PSA is significant in thatthe difference in the distribution of free PSA values and total PSAvalues has already been shown by Luderer to be resolvable into distinctpatient populations for CAP and BPD. A true measurement fornon-complexed PSA without over recovery can enhance the resolving powerof such assays, and thereby improve the diagnostic utility of PSAmeasurements in CAP diagnostics.

[0010] The present invention removes complexed PSA from furtherparticipation in a specific binding reaction, thereby removing anypossible interference due to the binding of complexed PSA in animmunoassay for free PSA. An added advantage of the present invention isthat the present method will measure non-complexed PSA while workingwith measurement antibodies that typically are used to measure eithertotal PSA or free PSA, unlike either the Lilja or Yeung methods whichrequire specific measurement antibodies to measure free PSA andcomplexed PSA, respectively. In particular, the present inventioninvolves either treating a fluid sample with a device so as to removephysically complexed PSA from the solution or treating a fluid samplewith an immuno-precipitating reagent so as to form an insoluble complexwhich contains either any ACT or any complexed PSA present in the samplebefore measuring for non-complexed PSA by means of an immunoassay. Oneshould note that the addition of the immuno-precipitating reagent canoccur either before the measurement immunoassay components are added tothe sample, or along with these components. In a preferred embodiment,the immuno-precipitating reagent can be part of the sample diluentcomponent used for generating a measurement signal. Also, if onephysically separates the precipitate from the sample, then conventionaltechniques can be used, such as washing, filtration, fluid transfer, andcentrifugation.

[0011] In the case of forming the insoluble complex, a specific bindingimmuno-precipitating reagent is added to a sample in a solution in anamount sufficient to bind and precipitate any ACT present in the sample,including PSA-ACT complex and ACT not complexed with PSA. Theimmuno-precipitating reagent binds to the ACT portion of the PSA-ACTcomplex but not to non-complexed PSA. The combination of PSA-ACT complexand the immuno-precipitating reagent is not able to bind to an antibodythat recognizes the PSA portion of the PSA-ACT complex for measurementby an immunoassay. Most importantly, the combination of either PSA-ACTcomplex or ACT and immuno-precipitating reagent is of a sufficient massto precipitate out of solution. (For the purposes of the presentinvention, “precipitate” includes any physical or chemical processwhereby PSA-ACT complex is transported from a dissolved state, (i.e., insolution), to an out of solution state that precludes participation in aspecific binding reaction involving antibodies.) Typically theimmuno-precipitating reagent is an anti-ACT antibody.

[0012] The present invention also includes using a multiple componentimmuno-precipitating reagent. For example, the immuno-precipitatingreagent can be comprised of two specific binding reagents. A firstspecific binding reagent binds to the ACT portion of the PSA-ACT complexbut does not have a sufficient mass when bound thereto to precipitatethe combination from the sample. A second specific binding reagent bindsto the bound first specific binding reagent, wherein the combined massof the PSA-ACT complex and the two specific binding reagents issufficient to form a precipitate. Neither of these specific bindingpartners interferes or binds to non-complexed PSA. Such antibodies arecommercially available from Scantibodies Laboratory, Inc. of Santee,Calif. USA. For example, a goat anti-ACT antibody can be used for thefirst specific binding partner, and a rabbit anti-goat antibody can beused for the second specific binding partner. The fluid sample is keptin contact with the second specific binding partner for a time and underconditions sufficient to bind all of the ACT and complexed PSA which isbound to the first specific binding partner, which are known to those ofskill in the art.

[0013] When using a pretreatment device, the method comprises foursteps. First, one contacts a biological fluid sample containing amixture of complexed PSA and non-complexed PSA with a pretreatmentdevice. The pretreatment device has attached to its surfaces an excessof at least one specific binding partner which specifically binds onlyto the ACT portion of complexed PSA and to ACT, but not to non-complexedPSA, leaving any non-complexed PSA unbound. Next, one keeps the fluidsample in contact with the pretreatment device for a time sufficient tobind all complexed PSA to any attached specific binding partners.Thirdly, one separates the fluid sample and the device. The fluid sampleis exposed to conventional specific binding assay reagents for detectingPSA under conditions which permit a measurement of PSA. Finally, onemeasures the amount of PSA present in the fluid sample, which isnon-complexed. Again, the present method will work with measurementantibodies that measure either total PSA or free PSA.

[0014] In one embodiment, a pretreatment device comprises two mainelements. The first is a filter that has a modified filter media. Thefilter is dimensioned and configured so as to permit the fluid sample toflow or be pulled through the filter media and into a vessel containingassay reagents. Suitable filter devices are commercially available. Thesecond element is that the modified filter media has attached to thesurface thereof at least one specific binding partner which specificallybinds only to the ACT portion of complexed PSA and to ACT, but not tonon-complexed PSA, leaving any non-complexed PSA unbound. The modifiedmedia has sufficient specific binding partner present to bind all ACTpresent in the sample, whether bound or otherwise.

[0015] In an alternative embodiment, the pretreatment device comprises aremovable media or solid support having at least one specific bindingpartner attached to the surface which specifically binds only to the ACTportion of complexed PSA and to ACT, but not to non-complexed PSA,leaving any non-complexed PSA unbound. The removable media can beconventional assay type media such as beads or strips. The removablemedia is dimensioned and configured so as to be placed into andwithdrawn from the fluid sample, which has been placed in a vessel.

[0016] In yet another alternative embodiment, the pretreatment devicecomprises a fluid sample vessel having the specific binding partnersdescribed above attached to the surface. The sample is placed within thevessel.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a diagrammatic view of an immunoassay for PSA.

[0018]FIG. 2 is a sectional view of a pretreatment filter device inaccordance with the present invention.

[0019]FIG. 3 is a sectional view of a removable pretreatment device inaccordance with the present invention.

[0020]FIG. 4 is a sectional view of a coated tube pretreatment device inaccordance with the present invention.

[0021]FIG. 5 is a diagrammatic view of an embodiment of the presentinvention wherein a precipitate is formed.

[0022]FIG. 6 is a graph showing the serial dilution of patient samplesusing the present invention to measure non-complexed PSA.

[0023]FIG. 7 is a graph showing the correlation between results achievedtesting samples from 63 patients with the present invention versus theLilja free PSA method.

BEST MODES FOR CARRYING OUT THE INVENTION PSA Assay

[0024] In preferred embodiments described below, the present methodcomprises measuring non-complexed PSA using any specific binding assaythat measures either total PSA or free PSA. Suitable PSA assays includea sandwich monoclonal/monoclonal immunoassay manufactured by TosohMedics, Inc. (Tosoh) of Foster City, Calif. USA for total PSA, one madeby Hybritech, Inc. of San Diego, Calif. USA for free PSA, or one made byCIS-Bio of Saclay, France for free PSA. FIG. 1 shows diagrammaticallyhow, in the final configuration in a traditional use, this assaycaptures PSA (10) using a capture antibody (12) and detects PSA using alabeled antibody (14).

Pretreatment Filter Device

[0025] As shown in FIG. 2, a pretreatment filter device comprises avessel (20) into which the fluid sample can be placed either by pouring,pipetting, or other conventional means for transferring fluids. One wallor surface of the vessel has an opening (22) for allowing the fluidsample to be removed from the pretreatment device by flowing or beingpulled by vacuum downward through the opening. Disposed within and aboutthe opening is a filter media (24) such as cellulose. Attached to thesurface of the filter media are a plurality of antibodies (14) which arespecific in binding ACT, either alone or complexed with PSA (16) but notnon-complexed PSA (18). Suitable antibodies include goat anti-ACT, whichare available from Scantibodies Laboratory, Inc. of Santee, Calif. USA.

[0026] These antibodies can be bound by conventional techniques known tothose of skill in the art. The filter media may be a bibulous materialcapable of drawing fluid sample into the media by capillary action. Thevolume of bibulous material present is sufficient to attach the excessof antibodies. Suitable bibulous materials include blotting papers,filter papers, non-woven natural polymers, and non-woven syntheticpolymers. The dimensions of the materials will vary depending uponsample size and absorptive capacity, as known to those of ordinary skillin the art.

Removable Pretreatment Device

[0027] As shown in FIG. 3, a removable pretreatment device can take theform of a disposable bead pack (30) which may be placed into a containeror vessel (32) either before or after placing a fluid sample into thevessel. The pack contains a plurality of beads (34) such as are used inexisting immunoassay devices. Suitable bead materials includepolystyrene or latex. Attached to the surface of the beads are an excessof antibodies (14) specific to ACT and the ACT portion of complexed PSA.Suitable antibodies include goat anti-ACT, which are available fromScantibodies Laboratory, Inc. of Santee, Calif. USA. These antibodiescan be bound by conventional techniques known to those of skill in theart.

[0028] The pretreatment device is placed into the sample for a time andunder conditions sufficient to allow any complexed PSA present to bindto the antibodies (14). Only non-complexed PSA (18) remains unbound inthe fluid sample. The pack may either be withdrawn from the fluid sampleand thrown away, left in place, or the sample may be removed leaving thepack in the vessel. Alternatively, a pretreatment device can be in theform of a coated dipstick or a disposable piece of coated media such ascellulose, nitrocellulose, glass fibers, and the like. One measures fornon-complexed PSA using conventional immunoassays for either total PSAor free PSA.

Coated Tube Pretreatment Device

[0029] As shown in FIG. 4, a coated tube pretreatment device comprises avessel (40) having disposed or deposited on the interior surfaces (42)an excess of anti-ACT antibodies (14). Suitable antibodies include goatanti-ACT, which are available from Scantibodies Laboratory, Inc. ofSantee, Calif. USA. These antibodies can be bound by conventionaltechniques known to those of skill in the art.

[0030] In use, ACT and complexed PSA (16) will bind to the anti-ACTantibodies, leaving non-complexed PSA (18) in the solution. One measuresfor non-complexed PSA using conventional immunoassays for either totalPSA or free PSA.

Immuno-Precipitating Reagent Method

[0031] As shown in FIG. 5, a fluid sample from a male human patient thatcontains non-complexed PSA (50), complexed PSA (52), and free ACT (54)is placed in a vessel. Typical sample sizes range from 25 μl to 5 ml.Immuno-precipitating reagent (56) is added to the sample, 25 μl to 5 mlof a goat anti-ACT commercially available from Scantibodies Laboratory,Inc. of Santee, Calif., USA. This amount is sufficient to bind andprecipitate the ACT present in the sample (58), either bound to PSA ornot. The immuno-precipitating reagent is allowed to remain in contactwith the sample for about one to five minutes. Non-complexed PSA ismeasured by a number of commercially available immunoassays for eithertotal PSA or free PSA.

Measurement of Non-Complexed PSA by Total PSA Immunoassay

[0032] Using the above pretreatment method, non-complexed PSA wasmeasured using a total PSA immunoassay from Scantibodies Laboratory,Inc. of Santee, Calif., USA. More particularly, 25 microliters ofpatient sample was pipetted into a plastic 12 mm by 75 mm tube. One ¼inch polystyrene bead coated with a monoclonal antibody to PSA, (acapture antibody), was added to each tube. Finally, 200 microliters ofbiotinylated goat anti-PSA was added to each tube. The tube wasincubated for two hours at room temperature (18 to 28 degrees C.). Thebead and tube were washed and liquid was removed from the tubecontaining the bead. To the tube was added 200 microliters ofhorseradish peroxidase conjugated streptavidin, which were allowed toincubate for 30 minutes. The tube and bead were washed, and all liquidwas removed. To develop a signal, 200 microliters of substrate(ortho-phenylenediamine) was added to each tube. The concentration ofPSA present, non-complexed PSA, was correlated with calibrators to thechange in color, which was quantitated spectrophotometrically. FIG. 6shows the sample dilution linearity for this method. Two samples werediluted and tested using the pretreatment and being measured fornon-complexed PSA by means of the Scantibodies Laboratory total PSAassay.

[0033] Using the above pretreatment method, non-complexed PSA ismeasured using a total PSA immunoassay from Tosoh. The manufacturer'sinstructions were followed for the assay of total PSA using their AIAtest method. For the generation of the non-complexed PSA results usingthe Tosoh total PSA assay, 100 microliters of patient sera was incubatedat room temperature with 100 microliters of Scantibodies Laboratory,Inc. goat anti-ACT for one hour. The mixture was treated as a normalpatient sample and assayed in accordance with the instructions forassaying total PSA by the AIA system, albeit non-complexed PSA resultswere obtained.

Measurement of Free PSA

[0034] For comparative purposes, free PSA is measured using a free PSAimmunoassay from CIS-Bio of Saclay, France. The manufacturer'sinstructions were followed for the assay of free PSA using the disclosedIRMA test methods.

Comparison of Measurement Methods

[0035] The following table compares results obtained for 63 patientsamples measured for free PSA (CIS-Bio free PSA immunoassay),non-complexed PSA (Tosoh Medics total PSA immunoassay and ScantibodiesLaboratory, Inc. immuno-precipitating reagent), and total PSA (TosohMedics total PSA immunoassay) as described above. All values are inng/ml. (FIG. 7 is a graph showing the correlation between resultsachieved testing samples from 63 patients with the present inventionversus the Lilja free PSA method.) FREE PSA NON-COMPLEXED PSA TOTAL PSA<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.2 <0.1 0.20.2 <0.1 0.3 0.2 0.1 0.3 0.3 0.2 0.3 0.3 0.3 0.3 0.2 <0.1 0.4 0.2 0.20.4 0.3 0.2 0.4 0.3 0.2 0.4 0.2 0.2 0.4 0.2 0.2 0.5 0.4 0.2 0.5 <0.1<0.1 0.6 0.2 0.1 0.6 0.1 <0.1 0.7 0.2 <0.1 0.7 0.2 0.2 0.7 0.5 0.4 0.80.5 0.4 0.8 0.6 0.5 0.9 0.3 0.1 1.0 0.4 0.3 1.0 0.5 0.1 1.1 0.5 0.5 1.20.5 0.3 1.3 0.6 0.6 1.3 0.3 0.2 1.4 0.4 0.1 1.5 0.8 0.7 1.8 0.7 0.5 2.11.1 0.9 2.1 0.2 <0.1 2.3 0.5 0.4 2.3 0.8 0.8 2.4 0.8 0.8 2.6 0.8 0.9 2.70.8 0.6 3.0 0.6 0.5 3.1 0.5 0.5 3.3 1.0 1.0 3.5 2.3 2.1 3.6 0.5 0.3 3.80.8 0.7 3.8 2.1 1.8 3.8 1.8 1.8 3.8 2.1 1.9 3.8 0.3 0.4 4.7 0.8 0.8 4.82.4 2.3 5.6 0.8 0.8 5.9 1.5 1.6 6.4 2.1 1.2* 7.7 2.2 2.0 8.4 0.6 0.711.9

[0036] The ordinarily skilled artisan can appreciate that the presentinvention can incorporate any number of the preferred features describedabove.

[0037] All publications or unpublished patent applications mentionedherein are hereby incorporated by reference thereto.

[0038] Other embodiments of the present invention are not presented herewhich are obvious to those of ordinary skill in the art, now or duringthe term of any patent issuing from this patent specification, and thus,are within the spirit and scope of the present invention.

I claim:
 1. A method for measuring the amount of non-complexed prostatespecific antigen in a sample from a male human patient comprising: a)adding a specific binding immuno-precipitating reagent to a sample in asolution in an amount sufficient to bind and precipitate anyα1-antichymotrypsin (ACT) present in the sample, whether complexed withprostate specific antigen (PSA), (PSA-ACT complex) or not; wherein theimmuno-precipitating reagent binds either to the ACT or the ACT portionof the PSA-ACT complex but not to non-complexed PSA, and the combinationof either ACT or PSA-ACT complex and the immuno-precipitating reagent isof a sufficient mass to precipitate out of solution; and b) measuringthe amount of non-complexed PSA, which remains in solution, by using anantibody that recognizes PSA in a specific binding reaction.
 2. Themethod of claim 1 wherein the antibody used for measuring non-complexedPSA in a specific binding reaction is a total PSA antibody.
 3. Themethod of claim 1 wherein the antibody used for measuring non-complexedPSA in a specific binding reaction is a free PSA antibody.
 4. The methodof claim 4 wherein the combination of immuno-precipitating reagent andPSA-ACT complex and immuno-precipitating reagent and ACT are separatedfrom the solution before measuring non-complexed PSA.
 5. The method ofclaim 1 wherein the precipitate is separated by means selected from thegroup consisting of washing, filtration, fluid transfer, andcentrifugation.
 6. The method of claim 1 wherein theimmuno-precipitating reagent is bound to a solid support.
 7. The methodof claim 6 wherein the solid support is colloidal.
 8. The method ofclaim 7 wherein the solid support is colloidal gold.
 9. The method ofclaim 1 wherein the immuno-precipitating reagent is selected from thegroup consisting of a polymerized antibody, conjugated antibodies,unpurified antibodies, fractionated antibodies, and purified antibodies.10. The method of claim 1 wherein the immuno-precipitating reagent isadded to the solution as part of an assay component for measuringnon-complexed PSA.
 11. The method of claim 1 wherein theimmuno-precipitating reagent is added substantially at the same time asthe non-complexed PSA is measured.
 12. The method of claim 1 wherein theimmuno-precipitating reagent is comprised of two specific bindingreagents; a first specific binding reagent that binds to the ACT portionof the PSA-ACT complex and ACT but does not have a sufficient mass whenbound thereto to cause either PSA-ACT complex or ACT to precipitate fromthe sample, and a second specific binding reagent that binds to thefirst specific binding reagent wherein the combined mass of either ACTor the PSA-ACT complex and the two specific binding reagents issufficient to form a precipitate; and the two specific binding reagentsare added to the sample before measuring the sample.
 13. The method ofclaim 12 wherein the immuno-precipitating reagent is added to thesolution as part of an assay component for measuring non-complexed PSA.14. The method of claim 12 wherein the immuno-precipitating reagent isadded substantially at the same time as the non-complexed PSA ismeasured.
 15. The method of claim 12 wherein the antibody used formeasuring non-complexed PSA in a specific binding reaction is a totalPSA antibody.
 16. The method of claim 12 wherein the antibody used formeasuring non-complexed PSA in a specific binding reaction is a free PSAantibody.
 17. A device for pretreating a biological sample that is to beassayed for non-complexed prostate specific antigen comprising: a) afilter having a modified filter media, the filter being dimensioned andconfigured so as to permit the fluid sample to flow or be pulled throughthe filter media and into a vessel containing assay reagents; and b) themodified filter media having attached to the surface thereof at leastone specific binding partner which binds to α1-antichymotrypsin (ACT),whether bound to prostate specific antigen (PSA) or not, but not tonon-complexed PSA, said specific binding partner being present in anamount sufficient to remove substantially all ACT present in the sample,whether bound or not.
 18. A device for pretreating a biological samplethat is to be assayed for non-complexed prostate specific antigencomprising a removable modified media or solid support, the removablemodified media having attached to the surface thereof at least onespecific binding partner which binds to α1-antichymotrypsin (ACT),whether bound to prostate specific antigen (PSA) or not, but not tonon-complexed PSA, said specific binding partner being present in anamount sufficient to remove substantially all ACT present in the sample,whether bound or not.
 19. A device for pretreating a biological samplethat is to be assayed for non-complexed prostate specific antigencomprising a vessel having attached to the surface thereof at least onespecific binding partner which binds to α1-antichymotrypsin (ACT),whether bound to prostate specific antigen (PSA) or not, but not tonon-complexed PSA, said specific binding partner being present in anamount sufficient to remove substantially all ACT present in the sample,whether bound or not.